JP2018521658A - タンパク質発現の選択的調節のための方法及び組成物 - Google Patents
タンパク質発現の選択的調節のための方法及び組成物 Download PDFInfo
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Abstract
Description
本出願は、参照によりその全体が本明細書に組み込まれている、2015年7月22日に出願された米国仮出願第62/195,546号の利益を主張する。
「MONS392WO_ST25.txt」というファイルに含まれる配列リストは、26.3キロバイト(オペレーティングシステムMS−Windowsで測定)であり、2016年6月28日に作成され、本明細書と共に提出され、参照により本明細書に組み込まれている。
発明の分野
本発明は、一般に、農業、植物育種及び分子生物学の分野に関する。より具体的には、本発明は、トランスジェニック植物の雄性生殖組織におけるタンパク質発現を選択的に調節するための方法及び組成物、並びにこれらの使用に関する。
ハイブリッド種子(雑種種子)は、密接に関連する植物のハイブリダイゼーション(雑種形成)または交雑受粉によって産生され、いずれかの親植物が所有していない形質の望ましい組み合わせを有する子孫ハイブリッド植物に生育させることができる。ハイブリッド植物は、植物の寸法、収量、栄養組成、病害抵抗性、除草剤耐性、ストレス耐性、気候適応、及び他の望ましい形質の改善などの優れた農業的特徴を示し得る。効率的なハイブリッド種子の生産には、植物自身の花粉が植物を自家受粉することが認められていないことが必要である。多くの作物のハイブリッド種子の生産を制限する主なものは、植物を雄性不稔性にし、自家受粉不能にする、単純で信頼性が高く経済的な方法の欠如である。
本発明は、一般に、トランスジェニック植物の雄性生殖組織におけるタンパク質発現を選択的に調節する方法、このような方法に有用な組換えDNA分子、ならびにこのような組換えDNA分子を含むトランスジェニック植物、細胞、及び種子に関する。本発明は、転写可能なポリヌクレオチド分子によってコードされたタンパク質の発現の改善された選択的調節を提供することができる雄性組織特異的siRNA(mts−siRNA)標的エレメントを提供することにより、当該技術を超えた改善を提供し、そしてこのようなmts−siRNA標的エレメントを含む組換えDNA分子及び組成物、及びこのようなmts−siRNA標的エレメントを、ハイブリッド種子の生産のためのトランスジェニック植物において雄性不稔性を誘発するために使用する方法を提供する。
配列番号:1−配列番号:17として本明細書で提供されるcDNA配列のヌクレオチド位置1429〜1628と95%の配列同一性を有するmts−siRNA標的エレメント配列。
本発明は、トランスジェニック植物の雄性生殖組織におけるタンパク質発現、例えば異種転写可能ポリヌクレオチド分子の発現、を選択的に調節するための、組換えDNA分子、組成物及び方法、並びにその使用を提供する。一態様では、本発明は、異種転写可能ポリヌクレオチドに作動可能に連結された雄性組織特異的低分子干渉RNA(mts−siRNA)標的エレメントを含む組換えDNA分子を提供する。このような組換えDNA分子は、トランスジェニック植物の雄性生殖組織における異種転写可能ポリヌクレオチドの発現を選択的に調節するために有用である。核酸配列は、規定されているように(当業者に知られているように、一方の開示は必然的に他方を定義する)、DNAまたはRNAとして提供することができる。さらに、所与の核酸配列の開示は、当業者に知られているように、必然的にその配列の相補体を定義し、包含する。
小分子RNAは、トウモロコシ房の4つの別個の成長段階及びトウモロコシ穂の3つの別々の成長段階から単離された。表1参照。房リッチの小分子RNAは、非常に早期の房発育段階(V7、V8/V9、V10/V11、及びV12)で単離された(図1参照)。これは、房リッチの小分子RNA配列を得るために従来技術で使用されていたものよりも若い雄性組織から小分子RNAを産生した。
mts−siRNA標的配列が豊富であると同定された6つのcDNA配列の領域に対応するDNA配列を用いて、組換えDNA構築物及び植物形質転換ベクターを作製した。組換えDNA構築物及び植物形質転換ベクターは、mts−siRNA標的エレメントに作動可能に連結された導入遺伝子の房特異的サイレンシングがmts−siRNA媒介サイレンシングを介して生じる、トランスジェニック植物の生産に有用であるように設計された。
組換えDNA構築物を含有する形質転換ベクターを、Agrobacteriumに基づく形質転換方法及びトウモロコシ未成熟胚及び当該技術分野で公知の技術とともに使用した。R0植物の葉の試料を収集し、分子分析を、組換えDNA構築物の単一コピー(1つの挿入物)または2重コピー(2つの独立した挿入物)を含み、ベクター骨格の存在しないトランスジェニック植物を選択するために行った。単一コピー事象はグリホサートを噴霧されず、自家受粉してR1種子収集に向けられ、一方2重コピーはR0グリホサート噴霧に使用して、栄養耐性を評価し、雄性不稔性を誘発した。グリホサートを噴霧されなかったトランスジェニック植物は、アレクサンドル染色及び顕微鏡観察によって決定される正常開花、正常花粉放出及び正常花粉発育を有していた。
トランスジェニック植物の房のCP4−EPSPSタンパク質の免疫局在化を用いて、細胞及び組織レベルでのタンパク質発現を分析して、作動可能に連結されたmts−siRNA標的エレメントの存在による花粉中のCP4−EPSPSタンパク質発現の喪失を確認した。配列番号:1に作動可能に連結されたcp4−epsps導入遺伝子または作動可能に連結されたmts−siRNA標的エレメントを有さないcp4−epsps導入遺伝子を含むR3世代のトランスジェニック植物も、温室内で生育させた。V2段階で1×グリホセート(0.75ポンドae/エーカー)を植物に噴霧し、栄養耐性を確認した。1cm〜17cmの房を、葯組織が小胞子母細胞に存在し、自由小胞期であるV8〜V12段階で収穫した。葯を、解剖用鉗子を用いて房小穂から取り出し、穏やかな真空下でリン酸緩衝食塩水(PBS)中の3.7%ホルムアルデヒドの中で直ちに固定した。PBSで洗浄した後、組織を包埋培地に入れ、直ちに凍結させた。凍結した組織ブロックを−80℃で保存したのち、−20℃のミクロトームで切片化し、帯電したスライド上に集めた。
ハイブリッド生産における最適な使用のためには、栄養性除草剤耐性(低作物害により測定される)と組み合わされて、除草剤の散布後のフィールド条件下における非常に低い葯押出しを有することが望ましい。ハイブリッドトウモロコシの生産の他の側面、例えば、植物の高さ及び収量も望ましい。これを評価するために、mts−siRNA標的エレメントに作動可能に連結されたcp4−epsps導入遺伝子を含むトランスジェニック植物を、フィールド条件下で高度な世代で試験し、多数のパラメータを測定した。
本発明のトランスジェニック植物及び種子は、ハイブリッド種子生産を含む育種目的で使用することができる。mts−siRNA標的エレメントに作動可能に連結されたグリホサート耐性EPSPSタンパク質をコードする導入遺伝子を含む組換えDNA構築物を含むトランスジェニックトウモロコシ植物は、田畑などの領域に植え付けられる。他の親トウモロコシ植物(複数可)は、同じ区域に存在しても存在しなくてもよい。種子生産中の雑草防除のために、グリホサートは、Roundup(登録商標)農産物ラベルに指示された栄養段階でトランスジェニックトウモロコシ植物に散布され得る。
Claims (22)
- 配列番号:1、2、5〜8、10〜12、16、23〜92及びこれらの相補体からなる群から選択される配列を含むmts−siRNA標的エレメントを含む組換えDNA分子であって、前記mts−siRNA標的エレメントが、異種の転写可能なポリヌクレオチド分子に作動可能に連結されている前記組換えDNA分子。
- 前記異種転写可能ポリヌクレオチド分子が、植物において除草剤耐性を付与するタンパク質をコードする請求項1に記載の組換えDNA分子。
- 前記異種転写可能なポリヌクレオチド分子が、グリホサート耐性5−エノールピルビルシキミ酸3−リン酸シンターゼ(EPSPS)をコードする請求項2に記載の組換えDNA分子。
- 前記mts−siRNA標的エレメントを異種の転写可能ポリヌクレオチド分子に作動可能に連結することを含む組換えDNA分子を産生する方法。
- 前記mts−siRNA標的エレメントが、配列番号:1、2、5〜8、10〜12、16、23〜92及びこれらの相補体からなる群から選択される配列を含む請求項4に記載の方法。
- 請求項1に記載の組換えDNA分子をそのゲノムに含むトランスジェニック植物またはその一部。
- 前記DNA分子を含む請求項6に記載のトランスジェニック植物の種子。
- 前記植物が単子葉植物である請求項6に記載の植物。
- 前記植物がトウモロコシ植物である請求項8に記載の植物。
- 前記トランスジェニック植物の雄性生殖組織におけるタンパク質の発現を選択的に調節する方法であって、前記トランスジェニック植物において、請求項1に記載の組換えDNA分子を発現させることを含む前記方法。
- 前記タンパク質が、グリホサート耐性5−エノールピルビルシキミ酸3−ホスフェートシンターゼ(EPSPS)を含む請求項10に記載の前記方法。
- トランスジェニック植物において雄性不稔性を誘発する方法であって、
a)配列番号:1、2、5〜8、10〜12、16、23〜92及びこれらの相補体からなる群から選択される配列を含むmts−siRNA標的エレメントであって、少なくとも第1の除草剤に対する耐性を付与するタンパク質をコードする異種転写可能ポリヌクレオチド分子に作動可能に連結されている前記mts−siRNA標的エレメントを含む組換えDNA分子を含むトランスジェニック植物を成長させること;及び
b)前記トランスジェニック植物に有効量の前記除草剤を散布し、この除草剤の散布が、前記トランスジェニック植物の雄性生殖組織の発育に先立って、またはそれと同時に行われ、これにより前記トランスジェニック植物に雄性不稔性を誘発すること;
を含む前記方法。 - 前記異種転写可能ポリヌクレオチド分子が、グリホサート耐性5−エノールピルビルシキミ酸3−リン酸シンターゼ(EPSPS)をコードする請求項12に記載の方法。
- 前記除草剤がグリホサートである請求項12に記載の方法。
- 前記除草剤の有効量が、1エーカーあたり約0.125ポンド酸当量〜1エーカー当たり約8ポンド酸当量のグリホサートである請求項14に記載の方法。
- 前記有効量の除草剤が、V4、V5、V6、V7、V8、V9、V10、V11、V12、V13及びV14段階からなる群から選択される発育段階に散布される請求項12に記載の方法。
- ハイブリッド種子を生産する方法であって、
a)配列番号:1、2、5〜8、10〜12、16、23〜92及びそれらの相補体からなる群より選択される配列を含むmts−siRNA標的エレメントを含む組換えDNA分子を含むトランスジェニック植物に、有効量の除草剤を散布し、且つ前記mts−siRNA標的エレメントが、少なくとも第一の除草剤に対する耐性を付与するタンパク質をコードする異種転写可能ポリヌクレオチド分子に作動可能に連結され、且つ前記除草剤の散布が、前記トランスジェニック植物の雄性生殖組織の発育に先立って、またはそれと同時に行われ、これにより前記トランスジェニック植物に雄性不稔性を誘発すること;
b)前記トランスジェニック植物を第2の植物からの花粉で受粉させること;及び
c)前記トランスジェニック植物からハイブリッド種子を形成させること;
を含む前記方法。 - 前記受粉することが、風媒受粉を起こさせることを含むことを特徴とする請求項17に記載の方法。
- 前記異種転写可能ポリヌクレオチド分子が、グリホサート耐性5−エノールピルビルシキミ酸3−リン酸シンターゼ(EPSPS)をコードする請求項17に記載の方法。
- 前記除草剤がグリホサートである請求項17に記載の方法。
- 前記グリホサートが、1エーカーあたり約0.125ポンド酸当量〜1エーカー当たり約8ポンド酸当量の有効量で、発育と同時に散布される請求項20に記載の方法。
- 請求項17に記載の方法によって生産されたハイブリッド種子であって、前記組換えDNA分子を含む前記ハイブリッド種子。
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US10704057B2 (en) | 2015-07-22 | 2020-07-07 | Monsanto Technology Llc | Methods and compositions for selective regulation of protein expression |
AU2019215434B2 (en) | 2018-02-02 | 2020-11-05 | Monsanto Technology Llc | Maize event MON87429 and methods of use thereof |
MY195997A (en) * | 2018-04-18 | 2023-02-27 | Kimagri Corp Sdn Bhd | Recombinant Gene |
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